Anthrax lethal toxin (LT) and edema toxin (ET) are made of three protein components: protective antigen (PA), lethal factor (LF) and edema factor (EF). PA is the cell binding moiety which binds to cellular receptors Tumor Endothelial Marker-8 (TEM8) and Capillary Morphogenesis Protein-2 (CMG2). After binding these cellular receptors, PA is proteolytically cleaved by ubiquitously expressed cell surface proteases, and forms an active oligomer which allows binding of the two enzymatic moieties, LF and EF, and their transport into the cell cytosol. LF cleaves several mitogen-activated protein kinase kinases (MEKs) and rodent inflammasome sensor Nlrp1. It is thought that the cleavage of the MEK substrates is responsible or required for host death. EF is an adenylate cyclase which converts ATP to cAMP. These two toxins are the major virulence determinants of anthrax and vaccine and therapeutic development against this disease primarily targets these proteins. In 2017 our laboratory continued work on multiple projects in both development of anti-toxin vaccines and therapeutics, as well as a better understanding of a time-to-treat for anthrax disease. Although anthrax can be treated with antibiotics, these drugs are not effective once sufficient quantities of the toxins have been secreted systemically and entered cells. Furthermore, antibodies which are the only currently approved non-antibiotic therapeutics for anthrax cannot reverse the effects of the endocytosed anthrax toxins and are not effective at later time points of infection. We are continuing our collaboration with Panthera BioPharma in identification and characterization of small molecular inhibitors for both LF and EF. Testing of novel LF inhibitors done during 2017 in our spore infection rodent model drew attention to issues in drug pharmacokinetics and cell permeability which guided the design of the next generation of modified LF inhibitors. Following successful testing that showed the new LF inhibitors to provide full protection in the anthrax spore challenge model, one candidate from the new panel of inhibitors was submitted in the application for clinical trial testing. In the same collaborative effort, a novel small molecule EF inhibitor was identified and shown to block the enzymatic activity of the toxin. Characterization of this and other EF inhibitors is underway as part of this long-term ongoing collaborative effort. One of the major issues in the use of anthrax animal models for testing of therapeutics is the selection of an ideal post-exposure time to treat. Because PA and LF are produced and secreted by the bacterium within minutes following germination of anthrax spores, and their kinetics in the blood has been characterized for most animal models, the field has now been converging on utilizing the first appearance of measurable PA as the time for initiation of treatment. However, none of the studies used to develop models for testing anthrax therapeutics, including those used to support the FDA approval of the current monoclonal antibodies for anthrax, have investigated the kinetics of intracellular cleavage of the MEK protein substrates relative to appearance of PA in circulation. PA molecules are taken up by cells almost as rapidly as they are produced and we have shown that PA is continually removed until receptors are depleted. Furthermore, we and others have demonstrated that LF activity can be measured in cells up to 2 weeks after entry. Thus, we hypothesized that by the time measurable amounts of PA appear in the bloodstream, sufficient quantities of LF and EF have been delivered to cells to cause irreversible damage, making the appearance of PA a poor time-to-treat trigger. We undertook a detailed study on the cleavage of MEK proteins during anthrax infection in the mouse model relative to circulatory PA and the ability of therapeutics to save mice from anthrax. Our studies suggest that the failure of many therapeutics against anthrax may be related to the selection of circulatory PA as a marker for treatment initiation. In a separate collaborative effort during 2017, we continued efforts on development of vaccines against anthrax. We developed a single recombinant vaccine that targets PA along with two antigens from Yersinia pestis, the causative agent of plague. The vaccine produced significant neutralizing titers after only two immunizations. This novel vaccine was tested in mice, rats and rabbits and conferred protection against both inhalational anthrax and pneumonic plague. This new vaccine is the first example of a single vaccine that is protective against two dangerous bacterial pathogens that are high on the list of possible bioterror agents.